Sphingolipids are found in all animal cell membranes and recently have been suggested to play important roles in a wide variety of cellular functions, including cell-cell interactions, cell growth and differentiation, transformation, and signal transduction. In order to better understand the roles of sphingolipids in these complex processes, it is necessary to elucidate mechanisms responsible for generating and maintaining the intracellular distribution of these molecules in cell membranes. In this application, five broad projects pertaining to the molecular mechanisms underlying sphingolipid synthesis, sorting, and transport in animal cells will be pursued: (i) We will study the mechanism of Golgi targeting of ceramide (a key intermediate in sphingolipid biosynthesis) using a novel collection of ceramide analogs in which the chain length of the sphingosine backbone is varied from 6 to 20 carbon atoms and will test several hypotheses for retention of ceramide at this organelle; (ii) We will study plasma membrane lipid organization using fluorescent sphingolipid analogs whose spectral properties allow both qualitative and quantitative assessments of their distribution and concentration within various compartments of the living cell. We will evaluate the ability of these molecules to form "microdomains" at the plasma membrane and study their potential to cluster in caveolae. We will also examine the internalization and "sorting" of these sphingolipid analogs into subpopulations of vesicles during the first seconds of endocytosis; (iii) We will carry out detailed molecular studies on glucosylceramide synthase, a key enzyme in glycosphingolipid biosynthesis, which we recently cloned from rat. Specifically, we will study oligomerization of the enzyme in the Golgi, perform site-directed mutagenesis and use deletion mutants to map the catalytic site and possible Golgi retention motifs. We will also study the localization of the enzyme at the EM level; (iv) Using a novel fluorescence screening assay which we recently developed, we will attempt to identify and sequence sphingomyelin synthase using a yeast complementation or rescue approach; and (v) We will study "lipid sorting" at the Golgi apparatus and further examine the mechanisms of sphingolipid transport from this organelle to the cell surface. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in cells and are fundamental to the development of rational treatments of membrane or cell surface related disease states.